Direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model.

In a previous study we showed that pneumococcal adherence to epithelial cells was enhanced by a preceding respiratory syncytial virus (RSV) infection. RSV-glycoproteins, expressed on the infected cell surface, may play a role in this enhanced pneumococcal binding, by acting as bacterial receptors. In the current study, it was attempted to analyze the capacity of pneumococci to interact directly with RSV virions. By flow-cytometry, a direct interaction between RSV and pneumococci could be detected. Heparin, an inhibitor of RSV infectivity that interacts with RSV protein-G, blocked RSV-pneumococcal binding, indicating that the latter interaction is indeed mediated by protein-G. RSV-pneumococcal complexes showed enhanced adherence to uninfected human epithelial cells, compared with pneumococcal adherence without bound RSV, and this enhancement was also blocked by heparin. In addition, the significance of these findings in vitro was explored in vivo in a murine model. Both mice that were pretreated with RSV at day 4 before pneumococcal challenge and mice infected with both agents simultaneously showed significantly higher levels of bacteraemia than controls. Simultaneous infection with both agents enhanced the development of pneumococcal bacteraemia most strongly. It was hypothesized that direct viral binding is another mechanism by which RSV can induce enhanced pneumococcal binding to epithelial cells, a phenomenon that is translated in vivo by a higher invasiveness of pneumococci when administered simultaneously with RSV to mice. Apparently, RSV acts in this process as a direct coupling particle between bacteria and uninfected epithelial cells, thereby increasing colonization by and enhancing invasiveness of pneumococci.

Enhanced adherence of Streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus.

In the present study, we analyzed the effect of a preceding respiratory syncytial virus (RSV) infection of human respiratory epithelial cells on the adherence of Streptococcus pneumoniae tested by means of a cytometric fluorescence assay. Adherence of clinically relevant pneumococcal serotypes 3, 9, 14, 18, 19, and 23 was studied using uninfected and RSV-infected monolayers. To this end, monolayers of both human nasopharyngeal cells (HEp-2) and pneumocyte type II cells (A549) were infected with RSV serotype A. Adherence to uninfected epithelial cells varied between pneumococcal serotypes. After RSV infection of the monolayers, all serotypes showed a strongly (2- to 10- fold) and significantly increased adherence when compared with adherence to uninfected monolayers. Enhanced adherence was observed with both cell lines. By fluorescence and scanning electron microscopy, we observed redistribution of pneumococcal adherence over the epithelial surface due to RSV infection, with dense bacterial accumulations near to epithelial syncytia.

A hemolytic assay for the estimation of functional mannose-binding lectin levels in human serum.

A simple assay was developed to estimate functional mannose-binding lectin (MBL) levels in serum based on the principle of yeast-induced bystander lysis of chicken erythrocytes (ChE). The assay is sensitive to inhibition by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (which allows alternative pathway activation), ethylene diamine tetraacetic acid (EDTA), mannose, N-acetylglucosamine and C1 esterase inhibitor (C1-INH), whereas it was not inhibited by galactose. A high-titer human anti-mannan antibody-containing serum with 0.06 microg MBL/ml gave a functional signal corresponding to 0.12 microg equivalents MBL/ml, indicating that anti-mannan antibodies are poorly hemolytic in the assay. The assay is well suited for the large-scale testing of patient samples for a functional MBL pathway of complement activation.